This year we are still participating in the many projects relating to the analysis of peptides and proteins that have been brought to our attention by researchers at NHLBI and NIH. The goal is still to use advanced mass spectrometric technology to study the biology of a particular system by obtaining information on the identity of proteins either through mass spectrometric sequencing or by simply carefully measuring the masses of a sufficient number of its peptides. This technique is unique in identifying post-translational modifications (PTMs) essential for protein/cellular functions. With the state-of-the-art Micromass QTOF3 mass spectrometer and other LC-MS and MALDI-TOF instruments, we were able to perform many experiments on biological samples, mostly in this protein and peptide class. In addition,we seek new chemical methods to enhance the search routines in order to increase their reliablity. In this year, we have developed a new reagent that greatly enhances the a1 ion obtained by MS/MS analysis. The suggestion is that we will be able to locate proteins by using only the two bits of information provided by this mass and the parent ion. In other work continued from last yaer, we have identified all of the sites of glycosylation on human and mouse zona pellucida as well as its N- and C- terminal amino acids along with its disulfide linkages. We have also identified a new processing of the important stromal-derived factor 1 alpha and beta by human plasma. We have also discovered several factors involved in the specific regulation of the adaptor protein complex AP-3 by ARF GAP protein AGAP1 with P. Randazzo using our cross linking methods worked out last year. We continue our interest in the regulation of retroviral protease dimerization through reversible oxidative modification and have published another article on this subject with the Davis group.We continue to spend a significant amount of time optimizing sensitivity and resolution of our QTOF3,its capillary LC and its communication with the mass spectrometer. A new automatic calibration device for the electrospray system is being evaluated. The new 2D-LC ProteomeX system from ThermoFinnigan is under investigation as a source of high throughput analysis.